Journal: Respiratory Research

Article Title: PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

doi: 10.1186/1465-9921-10-119

Figure Lengend Snippet: PDGF-Rα expression does not preclude SMAD-dependent TGFβ signaling in the lung . Lung tissue from PDGF-Rα-GFP mice aged P4 and P12 was stained for SMAD 2/3 followed by Alexa Fluor-568-conjugated secondary antibody (red, C) and PoPo 3 iodide nuclear counterstain (blue, A). The area of nuclear SMAD 2/3 was determined from merged images (D) in the nuclei cells that either express PDGF-Rα (yellow- red SMAD merged with green GFP) or do not express PDGF-Rα (magenta - red SMAD merged with blue pseudocolored PoPo3) cells. The inset in D (secondary antibody only) shows minimal levels of Alexa Fluor-568 staining in the absence of the primary antibody. Scale bars are 20 μm. Results are summarized in the column graph (E). The error bars represent standard deviations from the averages for each group. *, P < 0.001. To verify that nuclear translocation was TGFβ dependent, Mlg cells were treated with either vehicle or 0.125 ng/ml TGFβ (F) for 30 min and stained for SMAD 2/3 followed by 4', 6-diamidino-2-phenylindole (DAPI) nuclear counterstain. More SMAD 2/3 localized to the nuclei of TGFβ-treated cells.

Article Snippet: Rabbit polyclonal anti-SMAD 2/3 primary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Staining, Translocation Assay

Journal: Scientific Reports

Article Title: Profibrogenic effect of high-mobility group box protein-1 in human dermal fibroblasts and its excess in keloid tissues

doi: 10.1038/s41598-018-26501-6

Figure Lengend Snippet: HMGB1 induced TGF-β1, Smads, Erk 1/2, Akt, and NF-κb in HDFs. ( a – c ) Effects of HMGB1 on TGF-β1 signaling pathway in vitro . Significant increases in the expression levels of TGF-β1, Smad 2, and Smad 3 mRNA were observed in the HMGB1 (50 ng)-treated HDFs (** p < 0.01). ( d ) Effects of HMGB1 on TGF-β and phosphor-Smad 2/3 complex protein expressions. ( e , f ) TGF-β and phosphor-Smad 2/3 complex protein levels were significantly increased in the HMGB1 (100 ng)-treated HDFs (** p < 0.01). ( g ) Western blotting analysis in normal dermal spheroids. Increased expression of TGF-β and phosphor-Smad 2/3 complex protein were observed in normal dermal spheroids treated with HMGB1 (400 ng and 1000 ng). ( h ) The levels of Erk 1/2, Akt, and NF-κb protein were detected by western blotting in HDFs lysates treated with HMGB1 (50 ng and 100 ng). ( i to k ) Erk 1/2, Akt, and NF-κb protein levels in HMGB1 (100 ng)-treated HDFs were significantly increased by 1.5-, 2.0-, and 2.3-fold, respectively, versus non-treated HDFs (** p < 0.01).

Article Snippet: We used primary mouse anti-collagen type-I (monoclonal Abcam, Cambridge, UK), mouse anti-collagen type-III mAb (Sigma), rabbit anti-TGF-β1 mAb (Abcam), rabbit anti-phosphor-Smad 2/3 mAb (Cell Signaling Technology), and actin antibodies were used (mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: In Vitro, Expressing, Western Blot